There are numerous situations where it is of interest to isolate a specific class of cells or to remove a particular set of cells from a mixture of cells. Techniques which have been employed include fluorescence cell sorting, magnetic immunobeads, complement-mediated lysis, affinity chromatography, centrifugal elutriation and polystyrene panning of cells. Cells having substantial density differences, such as that between platelets and red cells can be grossly fractionated by gradient centrifugation methodologies. However, mammalian cells with equivalent densities, such as tumor cells, lymphocyte subsets, granulocytes, or stem cells, require some form of separation using molecular recognition of surface markers which correlate with their phenotype. Similarly, such molecular mechanisms are required to separate viruses and bacteria from one another in complex mixtures. Each of the aforementioned methods have serious drawbacks for many applications, where there is interest in isolation or removal of particular subsets of cells.
Disadvantages of fluorescence activated cell sorting for recovery of viable sorted cells are the slowness of the procedure, the fact that the isolated cells are coated with antibody, and the limited amounts of cells which can be obtained by the procedure.
With immunobeads, it is difficult to recover the cells from the beads after separation; the cells are frequently coated with antibody and magnetic beads, and distinct separations are only difficulty achieved. Complement mediated lysis is problematic for two reasons; first, depletion of target cells is incomplete, and second, non-target cells can be adversely affected by exposure to complement and the toxic by-products of target cell lysis. Affinity chromatography of cells using, for example, Sephadex G-10 coupled to antibody, suffers from poor recovery and inefficient depletion of target cells. Centrifugal elutriation is not capable of separating different phenotypic subpopulations of cells of like size.
The last methodology, panning developed by Wysocki and Sato, PNAS, 75:2844, (1978), utilizing passively absorbed antibody on polystyrene, is particularly inadequate. Only low recoveries can be achieved, and the process suffers from lack of specificity and contamination of the separated cells with antibody.
As the immune defense system becomes elucidated, it is increasingly evident that subsets of cells can have relatively narrow ranges of activities. Thus, subsets can be specialized for response to a particular disease, such as neoplasia, infection, viral or cellular, etc., response to transplants, and the like. It is therefore of great interest to be able to identify and purify these subsets of cells, not only to understand their action, but also to use these cells for prophylactic and therapeutic purposes. In order to achieve the desired results, it is necessary that substantially pure populations of the desired subset or subsets of cells can be obtained. Furthermore, the cells should be: (1) free of antibodies on their surface, (2) viable, (3) capable of fulfilling their normal function and (4) responsive to activation by biologicals in the same manner as normal cells in their normal environment.